Molecular Epidemiologic Typing Systems of Bacterial Pathogens: Current Issues and Perpectives

The epidemiologic typing of bacterial pathogens can be applied to answer a number of different questions: in case of outbreak, what is the extent and mode of transmission of epidemic clone(s )? In case of long-term surveillance, what is the prevalence over time and the geographic spread of epidemic and endemic clones in the population? A number of molecular typing methods can be used to classify bacteria based on genomic diversity into groups of closely-related isolates (presumed to arise from a common ancestor in the same chain of transmission) and divergent, epidemiologically-unrelated isolates (arising from independent sources of infection). Ribotyping, IS-RFLP fingerprinting, macrorestriction analysis of chromosomal DNA and PCR-fingerprinting using arbitrary sequence or repeat element primers are useful methods for outbreak investigations and regional surveillance. Library typing systems based on multilocus sequence-based analysis and strain-specific probe hybridization schemes are in development for the international surveillance of major pathogens like Mycobacterium tuberculosis. Accurate epidemiological interpretation of data obtained with molecular typing systems still requires additional research on the evolution rate of polymorphic loci in bacterial pathogens.

Identification of hepatitis C virus subtype 2c by sequencing analysis in patients from Córdoba, Argentina

In Argentina, most information on hepatitis C virus (HCV) genotype distribution comes from studies carried out in Buenos Aires (east province). In order to identify HCV subtypes in central Argentina, nucleotide sequencing of core region was performed in samples from 36 patients living in Córdoba, the second most populated province of Argentina. The sequence analysis identified subtype 2c as the most prevalent (50%), followed by subtype 1b (33%) and to a lesser extent by subtypes 1a (11%), 3a (3%) and 4a (3%). This is the first report of circulation of HCV subtype 2c in this region of Argentina and also such high prevalence has never been found before in the genotype distribution of South America.

Identification of uropathogenic Escherichia coli clonal group A (CgA) in hospitalised patients

This study provides the first description of healthcare-associated infections with Escherichia coli clonal group A (CgA) isolates in Latin America. Isolates were typed by enterobacterial repetitive intergenic consensus-PCR, pulsed-field gel electrophoresis, E. coli phylogenetic grouping, multilocus sequence typing and fimH single nucleotide polymorphism analysis. Out of 42 E. coli hospital isolates studied, three belonged to E. coli phylogenetic group D and ST69 and had fimH sequences identical to that of the CgA reference strain ATCC BAA-457. E. coli CgA is another potential source of resistant infections in hospitals.

Study of simple sequence repeat markers from coffee expressed sequences associated to leaf miner resistance

The objective of this work was to identify expressed simple sequence repeats (SSR) markers associated to leaf miner resistance in coffee progenies. Identification of SSR markers was accomplished by directed searches on the Brazilian Coffee Expressed Sequence Tags (EST) database. Sequence analysis of 32 selected SSR loci showed that 65% repeats are of tetra-, 21% of tri- and 14% of dinucleotides. Also, expressed SSR are localized frequently in the 5'-UTR of gene transcript. Moreover, most of the genes containing SSR are associated with defense mechanisms. Polymorphisms were analyzed in progenies segregating for resistance to the leaf miner and corresponding to advanced generations of a Coffea arabica x Coffea racemosa hybrid. Frequency of SSR alleles was 2.1 per locus. However, no polymorphism associated with leaf miner resistance was identified. These results suggest that marker-assisted selection in coffee breeding should be performed on the initial cross, in which genetic variability is still significant.

PCR amplification and sequence analyses of reverse transcriptase-like genes in Crinipellis perniciosa isolates

Reverse transcriptase (RT) sequence analysis is an important technique used to detect the presence of transposable elements in a genome. Putative RT sequences were analyzed in the genome of the pathogenic fungus C. perniciosa, the causal agent of witches' broom disease of cocoa. A 394 bp fragment was amplified from genomic DNA of different isolates of C. perniciosa belonging to C-, L-, and S-biotypes and collected from various geographical areas. The cleavage of PCR products with restriction enzymes and the sequencing of various RT fragments indicated the presence of several sequences showing transition events (G:C to A:T). Southern blot analysis revealed high copy numbers of RT signals, forming different patterns among C-, S-, and L-biotype isolates. Sequence comparisons of the predicted RT peptide indicate a close relationship with the RT protein from thegypsy family of LTR-retrotransposons. The possible role of these retrotransposons in generating genetic variability in the homothallic C. perniciosa is discussed.

Sequencing and promoter analysis of the nifENXorf3orf5fdxAnifQ operon from Azospirillum brasilense Sp7

A 40-kb DNA region containing the major cluster of nif genes has been isolated from the Azospirillum brasilense Sp7 genome. In this region three nif operons have been identified: nifHDKorf1Y, nifENXorf3orf5fdxAnifQ and orf2nifUSVorf4. The operons containing nifENX and nifUSV genes are separated from the structural nifHDKorf1Y operon by about 5 kb and 10 kb, respectively. The present study shows the sequence analysis of the 6045-bp DNA region containing the nifENX genes. The deduced amino acid sequences from the open reading frames were compared to the nif gene products of other diazotrophic bacteria and indicate the presence of seven ORFs, all reading in the same direction as that of the nifHDKorf1Y operon. Consensus sigma54 and NifA-binding sites are present only in the promoter region upstream of the nifE gene. This promoter is activated by NifA protein and is approximately two-times less active than the nifH promoter, as indicated by the ß-galactosidase assays. This result suggests the differential expression of the nif genes and their respective products in Azospirillum.

Antigenic and genetic characterization of the first rabies virus isolated from the bat Eumops perotis in Brazil

Although the main transmitters of rabies in Brazil are dogs and vampire bats, the role of other species such as insectivorous and frugivorous bats deserves special attention, as the rabies virus has been isolated from 36 bat species. This study describes the first isolation of the rabies virus from the insectivorous bat Eumops perotis. The infected animal was found in the city of Ribeirão Preto, São Paulo. The virus was identified by immunofluorescence antibody test (FAT) in central nervous system (CNS) samples, and the isolation was carried out in N2A cell culture and adult mice. The sample was submitted to antigenic typing using a panel of monoclonal antibodies (CDC/Atlanta/USA). The DNA sequence of the nucleoprotein gene located between nucleotides 102 and 1385 was aligned with homologous sequences from GenBank using the CLUSTAL/W method, and the alignment was used to build a neighbor-joining distance-based phylogenetic tree with the K-2-P model. CNS was negative by FAT, and only one mouse died after inoculation with a suspension from the bat's CNS. Antigenic typing gave a result that was not compatible with the patterns defined by the panel. Phylogenetic analysis showed that the virus isolated segregated into the same cluster related to other viruses isolated from insectivorous bats belonging to genus Nyctinomops ssp. (98.8% nucleotide identity with each other).

Molecular characterization of Cryptosporidium spp. from HIV infected patients from an urban area of Brazil

Cryptosporidium spp. are important cause of enteric disease in humans, but may also infect animals. This study describes the relative frequency of several Cryptosporidium species found in human specimens from HIV infected patients in the São Paulo municipality obtained from January to July 2007. Sequence analysis of the products of nested-PCR based on small subunit rRNA and Cryptosporidium oocyst wall protein coding genes revealed 17 (63.0%) isolates of C. hominis, four (14.8%) C. parvum, five (18.5%) C. felis and one (3.7%) C. canis. These findings suggest that, in urban environments of Brazil, the cat adapted C. felis may play a potential role in the zoonotic transmission of cryptosporidiosis whereas the anthroponotic transmission of cryptosporidiosis caused by C. hominis seems to predominate.

Refractory pemphigus vulgaris associated with herpes infection: case report and review

BACKGROUND: Pemphigus vulgaris (PV) is an autoimmune disease characterized by blistering of the skin and mucosa, which develops due to the interaction between predisposing genetic and environmental factors. Infections caused by members of the Herpesviridae family have been suggested as a possible triggering factor for PV. OBJECTIVE AND METHODS: In this report, we investigate the presence of herpesviruses in refractory lesions on the right upper eyelid. The lesion has persisted despite the treatment with corticosteroids. Polymerase chain reaction (PCR) and DNA sequence analysis have been used to detect the DNA of HSV 1/2, VZV, EBV, CMV, HHV-6, HHV-7, and HHV-8. RESULTS: The sample collected from the right upper eyelid has tested positive for HSV 1/2. Sequence analysis has confirmed the PCR results and allowed the identification of the HSV strain as belonging to type 1. After treatment with acyclovir, the lesion of the right upper eyelid has cleared and not relapsed. CONCLUSION: When patients present PV lesions which are refractory to corticosteroid therapy, herpetic infection should be considered.

TESTING A SUBTYPE-SPECIFIC GP41 AMPLIFICATION METHOD FOR GENOTYPING INDIVIDUALS INFECTED BY HUMAN IMMUNODEFICIENCY VIRUS TYPE-1 IN THE BRAZILIAN POPULATION OF ITAJAÍ, SOUTH BRAZIL

The method used by YAGYU et al. for the subtype-specific polymerase chain reaction (PCR) amplification of the gp41 transmembrane region of the human immunodeficiency virus type-1 (HIV-1) env gene, was tested. HIV-1 proviral DNA from 100 infected individuals in Itajaí, South Brazil was used to analyze this method. Seventy individuals were determined according to this method as having PCR products at the expected size for subtypes B, C, D and F. Of these individuals, 26 (37.1%) were observed as having the expected amplification for subtype C, and 42 (60%) were observed as having the expected products for subtypes B and D. Of the subtype B and D amplicons, 16 (22.9%) were classified as subtype D, and 26 (37.1%) were classified as subtype B. Two individuals (2.9%) had amplicons that were observed after subtype F-specific amplification was performed. Sequencing and comparing the patient sequences to reference sequences confirmed the classification of sequences of subtypes C and B. However, sequences that were falsely determined as being D and F in the PCR assay were determined as being subtypes C and B, respectively, by sequence analysis. For those individuals from whom no amplified products were obtained, a low viral load that was indicated in their patient history may explain the difficulty in subtyping by PCR methods. This issue was demonstrated by the results of ANOVA when testing the effect of viral load on the success of PCR amplification. The alignment of the obtained sequences with HIV-1 reference sequences demonstrated that there is high intra-subtype diversity. This indicates that the subtype-specific primer binding sites were not conserved or representative of the subtypes that are observed in the Brazilian populations, and that they did not allow the correct classification of HIV-1 subtypes. Therefore, the proposed method by YAGYU et al. is not applicable for the classification of Brazilian HIV-1 subtypes.

MOLECULAR CHARACTERIZATION OF INFLUENZA B VIRUS OUTBREAK ON A CRUISE SHIP IN BRAZIL 2012

In February 2012, an outbreak of respiratory illness occurred on the cruise ship MSC Armonia in Brazil. A 31-year-old female crew member was hospitalized with respiratory failure and subsequently died. To study the etiology of the respiratory illness, tissue taken at necropsy from the deceased woman and respiratory specimens from thirteen passengers and crew members with respiratory symptoms were analyzed. Influenza real-time RT-PCR assays were performed, and the full-length hemagglutinin (HA) gene of influenza-positive samples was sequenced. Influenza B virus was detected in samples from seven of the individuals, suggesting that it was the cause of this respiratory illness outbreak. The sequence analysis of the HA gene indicated that the virus was closely related to the B/Brisbane/60/2008-like virus, Victoria lineage, a virus contained in the 2011-12 influenza vaccine for the Southern Hemisphere. Since the recommended composition of the influenza vaccine for use during the 2013 season changed, an intensive surveillance of viruses circulating worldwide is crucial. Molecular analysis is an important tool to characterize the pathogen responsible for an outbreak such as this. In addition, laboratory disease surveillance contributes to the control measures for vaccine-preventable influenza.

Echinococcus granulosus sensu lato GENOTYPES IN DOMESTIC LIVESTOCK AND HUMANS IN GOLESTAN PROVINCE, IRAN

Cystic echinococcosis (CE) is a globally parasitic zoonosis caused by larval stages of Echinococcus granulosus. This study investigated E. granulosus genotypes isolated from livestock and humans in the Golestan province, northern Iran, southeast of the Caspian sea, using partial sequencing data of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1) mitochondrial genes. Seventy E. granulosus isolates were collected from animals in slaughterhouses: 18 isolates from sheep, 40 from cattle, nine from camels, two from buffaloes and one from a goat, along with four human isolates (formalin-fixed, paraffin-embedded tissues) from CE patients of provincial hospitals. All isolates were successfully analysed by PCR amplification and sequencing. The sequence analysis found four E. granulosus genotypes among the 74 CE isolates: G1 (78.3%), G2 (2.7%), G3 (15%) and G6 (4%). The G1-G3 complex genotype was found in all of the sheep, goat, cattle and buffalo isolates. Among the nine camel isolates, the frequency of G1-G3 and G6 genotypes were 66.7% and 33.3%, respectively. All four human CE isolates belonged to E. granulosus sensu stricto. This study reports the first occurrence of the G2 genotype in cattle from Iran and confirms the previously reported G3 genotype in camels in the same country.

Intrahospital spread of carbapenem-resistant Pseudomonas aeruginosa in a University Hospital in Florianópolis, Santa Catarina, Brazil

INTRODUCTION: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) has been isolated with increasing frequency in Brazilian hospitals. Since June 2003, its detection in a teaching hospital in the city of Florianópolis, Brazil, has increased. This study aimed to investigate the minimal inhibitory concentration (MIC), presence of Metallo-β-lactamase (MβL) and a possible clonal relationship among the isolates. METHODS: The study included 29 CRPA and seven isolates with reduced susceptibility. The MIC was determined by agar-dilution. Detection of MβL was performed by Double Disk Sinergism (DDS) and Combined Disk (CD). The MβL gene was verified by PCR and nucleotide sequence analysis. Epidemiological typing was performed by pulsed-field gel electrophoresis. RESULTS: Among the 29 carbapenem-resistant isolates, polymyxin B presented 100% susceptibility and piperacillin/tazobactam 96.7%. Seventeen (62%) strains were verified as clonal (A clone) and among these, six isolates indicated phenotypically positive tests for MβL and harbored the blaSPM-1 gene. The first CRPA isolates were unrelated to clone A, harbored blaIMP-16 and were phenotypically positive only by CD. CONCLUSIONS: The spread of a high-level of resistance clone suggests cross transmission as an important dissemination mechanism and has contributed to the increased rate of resistance to carbapenems. This study emphasizes the need for continuous surveillance and improved strategies.

Molecular detection of Trypanosoma sp. and Blastocrithidia sp. (Trypanosomatidae) in phlebotomine sand flies (Psychodidae) in the Federal District of Brazil

Abstract: INTRODUCTION : This study describes the occurrence of trypanosomatids in phlebotomines in Brasília, Brazil. METHODS : Two hundred and ten females of 13 sand fly species were analyzed by polymerase chain reaction (PCR) using different molecular markers (D7 24Sα rRNA, kDNA, and ITS1) and sequencing. RESULTS : PCR revealed trypanosomatid-positive samples from Nyssomyia whitmani and Evandromyia evandroi, which were negative by kDNA and ITS1 Leishmania-specific PCRs. DNA sequence analysis of D7 24Sα rRNA amplicons indicated the occurrence of Blastocrithidia sp. and Trypanosoma sp. in Nyssomyia whitmani and Evandromyia evandroi, respectively. CONCLUSIONS : Two trypanosomatid species other than Leishmania sp. were found to circulate in sand flies in Central Brazil.

An oligonucleotide probe derived from kDNA minirepeats is specific for Leishmania (Viannia)

Sequence analysis of Leishmania (Viannia) kDNA minicircles and analysis of multiple sequence alignments of the conserved region (minirepeats) of five distinct minicircles from L. (V.) braziliensis species with corresponding sequences derived from other dermotropic leishmanias indicated the presence of a sub-genus specific sequence. An oligonucleotide bearing this sequence was designed and used as a molecular probe, being able to recognize solely the sub-genus Viannia species in hybridization experiments. A dendrogram reflecting the homologies among the minirepeat sequences was constructed. Sequence clustering was obtained corresponding to the traditional classification based on similarity of biochemical, biological and parasitological characteristics of these Leishmania species, distinguishing the Old World dermotropic leishmanias, the New World dermotropic leishmanias of the sub-genus Leishmania and of the sub-genus Viannia.